Compositions and methods for promoting internalization and degradation of urokinase-type plasminogen activator

ABSTRACT

The invention includes compositions and methods for promoting internalization and degradation of urokinase-type plasminogen activator.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of international patent applicationPCT/US98/21800, filed on Oct. 15, 1998.

This application is entitled to priority pursuant to 35 U.S.C. §119(e)to U.S. provisional patent application No. 60/062,274, which was filedon Oct. 17, 1997.

FIELD OF THE INVENTION

The field of the invention is regulation of cell adhesion and migration.

BACKGROUND OF THE INVENTION

Plasminogen activators convert the inactive zymogen plasminogen into thebroad-spectrum proteolytic enzyme, plasmin (Higgins et al., 1990, Annu.Rev. Pharmacol. Toxicol. 30:91-121; Holden, 1990, Radiology174:993-1001; Mayer, 1990, Clin. Biochem. 23:197-211). One type ofplasminogen activator, designated urokinase-type plasminogen activator(uPA), is a component of the circulatory system and other fluidcompartments of the mammalian body.

uPA is the principal cell-associated plasminogen activator and has beenimplicated in several biological processes including angiogenesis,organogenesis, ovulation, inflammation, cancer, tumor cell invasion andmetastasis, atherosclerosis, and other biological and pathologicalprocesses characterized by cell migration through physiological barrierssuch as fibrin and basement membranes (Gyetko et al., 1994, J. Clin.Invest. 93:1380-1387; Gyetko et al., 1996, J. Clin. Invest.97:1818-1826; Shapiro et al., 1997, Am. J. Pathol. 150:359-369; Dado etal., 1994, Fibrinolysis 8(Suppl. 1):189-203).

uPA is synthesized as a single chain zymogen, designated single chainuPA (scuPA), which exhibits little urokinase activity (Ellis et al.,1987, J. Biol. Chem. 262:14998-15003; Petersen et al., 1988, J. Biol.Chem. 263:11189-11195; Husain, 1991, Biochemistry 30:5707-5805; Colleenet al., 1986, J. Biol. Chem. 261:1259-1266). Activation of scuPA occursby enzymatic cleavage of scuPA, yielding two-chain uPA (tcuPA).Physiological formation of tcuPA from scuPA is catalyzed primarily byplasmin (Robbins et al., 1967, J. Biol. Chem. 242:2333-2342). scuPA mayalso be activated by binding of scuPA to the cell-surface receptor,uPAR. In the case of scuPA binding to uPAR, scuPA remains a single chainmolecule, but is active (Higazi et al., 1995, J. Biol. Chem.270:17375-17380).

The activity of uPA is regulated, in part, by plasminogen activatorinhibitor-1 (PAI-1), which is a member of the serine protease inhibitor(SERPIN) family of proteins (Kruithof, 1988, Enzyme 40:113-121; Potempaet al., 1994, J. Biol. Chem. 269:15957-15960; Lijnen et al., 1994, Eur.J. Biochem. 224:567-574). PAI-1 is thought to be the most relevantinhibitor of uPA activity in the fluid phase, due to its high secondorder rate constant of inhibition, 1.7×10⁻⁸ M⁻¹.s⁻¹, which is higherthan any other protease inhibitor(Hekman et al., 1988, Arch. Biochem.Biophys. 262:199-210).

A soluble recombinant form of uPAR, designated suPAR, is known anddiffers from uPAR by lacking the portion of uPAR that links the receptorto the cell surface. suPAR possesses the same properties as uPAR withrespect to binding and activating scuPA and promoting the adhesivity ofthe uPA-uPAR complex (Higazi et al., 1995, J. Biol. Chem.270:17375-17380; Higazi et al., 1996, Blood 87:3545-3549).

Binding of scuPA to uPAR enhances urokinase activity of scuPA (Higazi etal., 1995, J. Biol. Chem. 270:17375-17380). Formation of the scuPA-uPARcomplex also dampens the capacity of PAI-1 to inhibit scuPA activity,relative to the capacity of PAI-1 to inhibit tcuPA activity (Higazi etal., 1996, Blood 87:3545-3549). Formation of a complex between scuPA anduPAR also alters the regulation of scuPA enzymatic activity by peptidesubstrates of plasmin and promotes binding of scuPA to vitronectin(Higazi et al., 1996, Thromb. Res. 84:243-252; Higazi et al., 1996,Blood 88:542-551; Wei et al., 1994, J. Biol. Chem. 269:32380-32388; Weiet al., 1996, Science 273:1551-1555; Deng et al., 1996, J. Cell Biol.134:1563-1571; Stefansson et al., 1996, Nature 383:441-443; O'Reilly etal., 1996, Nature Med. 2:689-692).

A region of uPA, comprising the protein sequence RHRGGS (SEQ ID NO:1) atamino acid positions 179-184, is required for inhibition of uPA activityby PAI-1 (Madison et al., 1990, J. Biol. Chem. 265:21423-21426).Conservation of this sequence among mammalian uPA proteins has beendemonstrated (Adams et al., 1981, J. Biol. Chem. 266:8476-8482). Workingwith a different plasminogen activator protein, namely tissue-typeplasminogen activator (tPA), Madison et al. have identified a region ofPAI-1 which is involved in inhibition of tPA by PAI-1 (1990, J. Biol.Chem. 265:21423-21426). This region of PAI-1 comprises the sequenceRMAPEEIIMDR (SEQ ID NO:2) at amino acids 346-356. It has been postulatedthat electrostatic interactions between this region of PAI-1 and tPAplay a role in stabilizing a tPA-PAI-1 complex. Similarly, it has beenpostulated that electrostatic interactions between a region of PAI-1 anduPA may contribute to formation of a PAI-1-uPA complex. It has beenobserved, however, that the scuPA-uPAR complex is less susceptible toinhibition by PAI-1 (Higazi et al., 1996, Blood 87:3545-3549) than istcuPA or uPAR-bound tcuPA (Higazi et al., 1996, Blood 87:3545-3549;Ellis et al., 1990, J. Biol. Chem. 265:9904-9908).

In addition to inhibiting urokinase activity of uPA, PAI-1 also promotesthe internalization and lysosomal degradation of uPA, which involves theα₂-macroglobulin receptor/low density lipoprotein-related receptorprotein (α₂MR/LRP; Nykjaer et al., 1994, J. Biol. Chem.269:25668-25676). The complex formed between PAI-1 and tcuPA binds toα₂MR/LRP with considerably higher affinity than does either componentalone. Although it has been demonstrated that the increased affinity ofthe complex results from an independent contribution of epitopes presentin each ligand, a possible conformation-altering effect of PAI-1 uponuPA has not been excluded (Nykjaer et al., 1994, J. Biol. Chem.269:25668-25676).

When scuPA is bound to uPAR, scuPA is protected from inactivation byPAI-1. Furthermore, binding of scuPA to uPAR inhibits binding of scuPAto α₂MR/LRP and internalization of scuPA caused by such binding (Nykjaeret al., 1994, J. Biol. Chem. 269:25668-25676; Higazi et al., 1996, Blood87:3545-3549). Two mechanisms have been postulated for the reducedaffinity of uPAR-bound scuPA for α₂MR/LRP. Nykjaer et al. (supra)proposed that the site at which scuPA contacts α₂MR/LRP is shielded byuPAR. An alternative mechanism is that binding of scuPA to uPAR inducesa conformational change that both promotes scuPA binding to integrinligands and leads to a loss of the scuPA epitope recognized by α₂MR/LRP(Higazi et al., 1996, Blood 88:542-551). The latter proposed mechanismis consistent with the observation that soluble scuPA has a higheraffinity for α₂MR/LRP than does tcuPA and with the observation thattcuPA loses affinity for α₂MR/LRP when the active site of tcuPA isoccupied by diisofluoryl phosphate (Nykjaer et al., 1994, J. Biol. Chem.269:25668-25676).

scuPA bound to uPAR is active, protected from inactivation by PAI-1, andprotected from clearance from the cell surface mediated by binding ofscuPA to α₂MR/LRP and subsequent degradation. Furthermore, scuPA thatdissociates from uPAR reverts to an inactive conformation and becomesessentially insusceptible to inactivation by PAI-1. Thus, unbound scuPAretains the capacity to rebind to uPAR and revert once again to itsactive conformation.

There are abundant epidemiological data which indicate that theexpression or uPA and uPAR in human tissue correlates with theconversion of cells from a benign to a neoplastic state. Furthermore,expression of uPA and uPAR is associated with a wide variety of commonmalignancies, and is predictive of future development of thosemalignancies. Interference with uPA activity by binding an antibody touPA, by expression of an antisense oligonucleotide complementary to mRNAencoding uPA, or by overexpression of catalytically inactive forms ofuPA impede tumor progression in several experimental murine models ofhuman cancers (Ossowski, 1988, J. Cell Biol. 107:2437-2445; Ossowski etal., 1991, Canc. Res. 51:275-281; Kook et al., 1994, EMBO J.13:3983-3991; Crowley et al., 1993, Proc. Natl. Acad. Sci. USA90:5021-5025; Jankun et al., 1997, Canc. Res. 57:559-563).

The capacity to regulate uPA activity would enable the practitioner toregulate a number of important human diseases and symptoms thereof.There remains a significant unmet need for compositions useful formodulating the activity of uPA in a mammal, particularly in a human, andfor methods of using those compositions to treat pathological conditionsattributable to undesirable uPA activity in the mammal. Particularlyneeded are compositions and methods for promoting internalization anddegradation of scuPA which act independently of activation of scuPA byplasmin, independently of binding of scuPA to uPAR, and independently ofinactivation of soluble or uPAR-bound scuPA by PAI-1.

SUMMARY OF THE INVENTION

The invention includes a composition comprising a peptide having theamino acid sequence X₁X₂X₃X₄X₅X₆X₇X₈, wherein:

X₁ is hydrogen, an amino-terminal blocking group, or one to twenty aminoacid. residues;

X₂ is an amino acid selected from the group consisting of D, E, H, K,and R;

X₃ is an amino acid selected from the group consisting of E and D;

X₄ is an amino acid selected from the group consisting of I, L, and V;

X₅ is an amino acid selected from the group consisting of I, L, and V;

X₆ is an amino acid selected from the group consisting of M;

X₇ is an amino acid selected from the group consisting of D, E, H, K,and R; and

X₈ is hydrogen, a carboxyl-terminal blocking group, or one to twentyamino acid residues.

In one aspect,

X₁ is hydrogen or an amino-terminal blocking group;

X₂ is an amino acid selected from the group consisting of D, E, and R;

X₃ is an amino acid selected from the group consisting of D and E;

X₄ is I;

X₅ is I;

X₆ is M;

X₇ is an amino acid selected from the group consisting of D and E; and

X₈ is hydrogen or a carboxyl-terminal blocking group.

In a preferred embodiment,

X₁ is hydrogen;

X₂ is E;

X₃ is E;

X₄ is I;

X₅ is I;

X₆ is M;

X₇is D; and

X₈ is hydrogen.

In another aspect, the composition of the invention further comprising apharmaceutically acceptable carrier.

Also included in the invention is a method of affecting a biologicalprocess characterized by abnormal cell migration through a physiologicalbarrier. The method comprises administering the composition of theinvention to a mammal experiencing the biological process in an amountto affect the biological process.

In one aspect, the biological process is selected from the groupconsisting of angiogenesis, organogenesis, ovulation, inflammation,cancer, tumor cell invasion and metastasis, and atherosclerosis.

In a preferred embodiment, the mammal is a human.

The invention further includes a method of inhibiting PAI-1-dependentadhesion of a cell to a tissue of a mammal, the method comprisingadministering to the tissue the composition of the invention in anamount to inhibit adhesion of the cell to the tissue.

In one aspect, the tissue is in vivo in the mammal.

In a preferred embodiment, the mammal is a human.

Also included in the invention is a method of promoting clearance ofscuPA from the surface of a mammalian cell, the method comprisingadministering the composition of claim 1 to the cell in an amount topromote clearance of the scuPA from the cell.

In one aspect, the cell is a human cell.

In a preferred embodiment, the composition is administered in vivo inthe human.

Additionally, the invention includes a method of impeding pathologicalmigration of a cell in a mammal. The method comprises administering tothe mammal the composition of the invention in an amount effective toimpede pathological migration of the cell.

In one aspect, the composition is administered to the mammal at the siteof a tumor in the mammal.

In a preferred embodiment, the mammal is a human.

The invention yet further includes a method of inhibiting PAI-1 activityin a tissue of a mammal. The method comprises administering to thetissue the composition of the invention in an amount effective toinhibit PAI-1 activity in the tissue.

In a preferred embodiment, the mammal is a human.

In another preferred embodiment, the composition is administered in vivoin the human.

The invention also includes a kit comprising a peptide having the aminoacid sequence X₁X₂X₃X₄X₅X₆X₇X₈, wherein:

X₁ is hydrogen, an amino-terminal blocking group, or one to twenty aminoacid residues;

X₂ is an amino acid selected from the group consisting of D, E, H, K,and R;

X₃ is an amino acid selected from the group consisting of E and D;

X₄ is an amino acid selected from the group consisting of I, L, and V;

X₅ is an amino acid selected from the group consisting of I, L, and V;

X₆ is an amino acid selected from the group consisting of M;

X₇ is an amino acid selected from the group consisting of D, E, H, K,and R; and

X₈ is hydrogen, a carboxyl-terminal blocking group, or one to twentyamino acid residues, and an instructional material for using the kit.

Also included is a composition comprising a combination of a peptidehaving the amino acid sequence X₁X₂X₃X₄X₅X₆X₇X₈, wherein:

X₁ is hydrogen, an amino-terminal blocking group, or one to twenty aminoacid residues;

X₂ is an amino acid selected from the group consisting of D, E, H, K,and R;

X₃ is an amino acid selected from the group consisting of E and D;

X₄ is an amino acid selected from the group consisting of I, L, and V;

X₅ is an amino acid selected from the group consisting of I, L, and V;

X₆ is an amino acid selected from the group consisting of M;

X₇ is an amino acid selected from the group consisting of D, E, H, K,and R; and

X₈ is hydrogen, a carboxyl-terminal blocking group, or one to twentyamino acid residues, and

a thrombolytic agent.

In one aspect, the thrombolytic agent is selected from the groupconsisting of tissue plasminogen activator, streptokinase, urokinase,the streptokinase derivative and staphylokinase.

Further included in the invention is a composition comprising acombination of a peptide having the amino acid sequenceX₁X₂X₃X₄X₅X₆X₇X₈, wherein:

X₁ is hydrogen, an amino-terminal blocking group, or one to twenty aminoacid residues;

X₂ is an amino acid selected from the group consisting of D, E, H, K,and R;

X₃ is an amino acid selected from the group consisting of E and D;

X₄ is an amino acid selected from the group consisting of E, L, and V;

X₅ is an amino acid selected from the group consisting of I, L, and V;

X₆ is an amino acid selected from the group consisting of M;

X₇ is an amino acid selected from the group consisting of D, E, H, K,and R; and

X₈ is hydrogen, a carboxyl-terminal blocking group, or one to twentyamino acid residues, and

an anti-coagulating agent.

In one aspect, the anti-coagulating agent is selected from the groupconsisting of an agent which inhibits platelet function, and agent whichinhibits the activity of thrombin, and agent which promotes the activityof activated protein kinase C, an anti-thrombin III agent, and a tissuefactor pathway inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, comprising FIGS. 1A and 1B, is a pair of graphs which depict theeffects which individual peptides had on binding of scuPA to LM-TK⁻cells (α2MR⁺/LRP, uPAR⁻), wherein each individual peptide had a sequenceselected from the group consisting of EEIIMD (SEQ ID NO:3), REIIMD (SEQID NO:4), and EEIIMR (SEQ ID NO:5). In FIG. 1A, the effect of thepresence of the indicated peptide is shown, wherein “No Addition” meansthat none of the three peptides was added to the reaction mixture. InFIG. 1B, the effect of the presence of the indicated concentration ofpeptide EEIIMD is shown.

FIG. 2 is a bar graph which depicts the effect of peptide EEIIMD onbinding of the scuPA in the presence or absence of PAI-1 to LM-TK⁻cells.

FIG. 3 is a bar graph which depicts the effect of PAI-1 and the effectof peptide EEIIMD (SEQ ID NO:3) on the binding of the scuPA-suPAR andthe tcuPA-suPAR complexes to LM-TK⁻ cells.

FIG. 4 is a graph which depicts the effect of the presence of peptideEEIIMD (SEQ ID NO: 3) on the activity of PAI-1.

FIG. 5, comprising FIGS. 5A and 5B, is a pair of bar graphs which depictthe effects of the presence of peptide EEIIMD (SEQ ID NO: 3) on bindingof scuPA to α₂MR/LRP and to LM-TK⁻ cells. The results depicted in FIG.5A were obtained by assessing binding of labeled scuPA to purifiedα₂MR/LRP in the absence or presence of rRAP and in the absence orpresence of the peptide. Open boxes represent data collected in theabsence of rRAP, and striped boxes represent data collected in thepresence of rRAP. The data depicted in FIG. 5B were obtained byassessing binding of labeled scuPA to LM-TK⁻ cells in the absence orpresence of the peptide. Data representing binding in the presence of 4millimolar Ca²⁺ are represented by open boxes. Data representing bindingin the presence of 400 nanomolar rRAP are indicated by grey boxes. Datarepresenting binding in the presence of 100 micrograms/milliliteranti-α₂MR/LRP IgG are indicated by black boxes.

FIG. 6 is a bar graph which depicts the effect of the presence ofpeptide EEIIMD (SEQ ID NO: 3) on the internalization and degradation ofscuPA by LM-TK⁻ cells. “Control” refers to data collected in experimentswherein cells were incubated in the absence of rRAP and anti-α₂MR/LRP.“rRAP” refers to data collected in experiments wherein cells wereincubated in the presence of rRAP. “Ab” refers to data collected inexperiments wherein cells were incubated in the presence of ananti-α₂MR/LRP antibody. Data collected in experiments performed in theabsence of the peptide are indicated by open boxes. Data collected inexperiments performed in the presence of the peptide are indicated bystriped boxes.

FIG. 7 is a graph depicting a comparison of the distribution of labeledfibrin micro-emboli and radiolabeled fibrinogen.

FIG. 8 is a graph depicting clearance of micro-emboli from the lungs ofwild type animals and various animals having deletion mutations intissue-type plasminogen activator (tPA^(−/−)), urokinase-typeplasminogen activator (uPA^(−/−)), and the urokinase receptor(uPAR^(−/−)).

FIG. 9 is a graph depicting data obtained when the phenotype of theurokinase-type plasminogen activator (uPA^(−/−)) mice was rescued byinfusion of two chain uPA. Clot lysis in the uPA^(−/−) mice wascomplete.

DETAILED DESCRIPTION

The invention is based on the discovery of a peptide which promotesbinding of scuPA to LM-TK⁻ cells and thereby promotes internalizationand degradation of scuPA by those cells. The peptide of the inventionalso promotes binding of scuPA to purified α2MR/LRP. Hence the peptideof the invention is useful for affecting the physiological availabilityof scuPA, and is thus useful for affecting biological processescharacterized by abnormal cell migration through a physiologicalbarrier, such biological processes including, but not being limited to,angiogenesis, organogenesis, ovulation, inflammation, cancer, tumor cellinvasion and metastasis, and atherosclerosis.

Internalization and degradation of cell-associated uPA mediated bybinding of uPA to α₂MR/LRP represents an important step in physiologicalcontrol of plasmin formation. Complexes formed between tcuPA and PAI-1are rapidly degraded. In contrast, the insusceptibility of scuPA toPAI-1 prevents clearance by PAI-1 inactivation prevents clearance ofscuPA from cell surfaces from occurring to any appreciable degree. Theseobservations suggest that α₂MR/LRP requires the presence of specificepitopes in scuPA for effective internalization and degradation thereof.

According to the data presented herein, the binding of scuPA to LM-TK⁻cells, which comprise α₂MR/LRP but lack uPAR, was stimulated by thepresence in the extracellular medium of the hexapeptide, EEIIMD (SEQ IDNO:3). The presence of peptide EEIIMD increased the value of the B_(max)constant for scuPA binding four-fold, and half-maximal effect wasachieved at a peptide concentration of about 50 micromolar. B_(max)represents the maximum number of binding sites of the surface of a cellat saturation. The magnitude of the increase in the maximum bindingvelocity was dependent on the charge of the C-terminal amino acid, butwas not dependent on the charge of the N-terminal amino acid of thepeptide.

Peptide EEIIMD (SEQ ID NO:3) promoted binding of scuPA to purifiedα₂MR/LRP. Peptide EEIIMD also accelerated the rate of internalizationand degradation of scuPA by LM-TK⁻ cells. Binding of scuPA to LM-TK⁻cells and internalization of scuPA by those cells in the presence ofpeptide EEIIMD were inhibited by the presence of either rRAP or ananti-α₂MR/LRP antibody. Peptide EEIIMD had no effect on binding of tcuPAto either LM-TK⁻ cells or purified α₂MR/LRP, and did not affectscuPA-uPAR complex formation. Thus, peptide EEIIMD regulates binding ofscuPA to α₂MR/LRP, and EEIIMD-promoted binding of scuPA to α₂MR/LRPrepresents a method of promoting internalization and degradation ofscuPA by cells comprising α₂MR/LRP independently of both activation ofscuPA by plasmin and binding of scuPA to uPAR. Promoting internalizationand degradation of scuPA by cells inhibits biological processescharacterized by abnormal cell migration through a physiological barrier

The presence of cell-associated scuPA was regulated by exposing LM-TK⁻cells to peptide EEIIMD, the sequence (SEQ ID NO:3) of which ishomologous with a portion of the amino acid sequence of PAI-1, beingamino acids 350-355 of PAI-1. Peptide EEIIMD was observed to be acompetitive inhibitor of PAI-1 binding to tcuPA. This peptide also boundto scuPA under experimental conditions under which PAI-1 did not bind toscuPA. It was furthermore observed that peptide EEIIMD promotedinternalization and degradation of scuPA by means of binding of scuPA tocellular α₂MR/LRP.

Thus, the peptide of the invention as described herein including, butnot limited to, peptide EEIIMD (SEQ ID NO:3) and peptides andpeptidomimetics derived therefrom, may be used to promote clearance ofscuPA from cell surfaces, thereby impeding pathologic cell migration. Inaddition, the peptide of the invention may be used synergistically withinhibitors of uPA-uPAR complex formation. Inhibitors of uPA-uPAR complexformation are known in the art and include those that are peptide- orantibody-based, those which are directed to the binding sequences ineither uPA or uPAR, those based on sequences from other proteinsbelieved to interact with the uPA-uPAR complex, organic inhibitors,antisense-based inhibitors, and the like. For example, Suramin may beused to inhibit complex formation. When inhibitors of uPA-uPAR complexformation are used alone in a subject experiencing a biological processcharacterized by abnormal cell migration through a physiologicalbarrier, a biologically relevant amount of scuPA remains bound to uPARor in the cellular environment near uPAR. A biologically relevant amountof scuPA may furthermore be capable of binding to uPAR owing topersistent local synthesis of scuPA. A biologically relevant amount ofscuPA means an amount of scuPA which is capable of influencing themigration of a cell through a physiological barrier. Thus, use of aninhibitor of uPA-uPAR complex formation alone is insufficient to resultin complete clearance of scuPA from a cell surface, such that abiologically relevant amount of scuPA does not remain on the cellsurface.

A peptide of the invention, when used alone or in combination with aninhibitor of uPA-uPAR complex formation, may be used to efficientlypromote clearance of scuPA from cell surfaces. When a peptide of theinvention and an inhibitor of uPA-uPAR complex formation are used incombination, a synergistic effect results, the peptide of the inventioncausing internalization of scuPA by means of scuPA binding to α₂MR/LRPand subsequent internalization and degradation of scuPA, and theuPA-uPAR binding inhibitor preventing sequestration of a biologicallyrelevant amount of scuPA bound to uPAR.

The peptide of the invention including, but not limited to, peptideEEIIMD (SEQ ID NO:3) is also useful as an inhibitor of PAI-1 activity.There is abundant human epidemiologic and experimental data which linkPAI-1 activity with thrombosis. As one example, arterial clots arerelatively resistant to thrombolytic agents, in part because thrombinbinding to fibrin promotes the release of PAI-1 by activated plateletswhich become trapped in the fibrin meshwork. Another important recentobservation is that PAI-1 regulates cell adhesion mediated by theuPA-uPAR complex. The peptide of the invention including, but notlimited to peptide EEIIMD and peptides and peptidomimetics derivedtherefrom, is a competitive inhibitor of PAI-1-binding to scuPA and totcuPA. The presence of the peptide of the invention in the extracellularmilieu inhibits cleavage of cell-surface scuPA by PAI-1 by removingscuPA so that it cannot be converted to tcuPA. Thus, the peptide of theinvention promotes endogenous thrombolysis, reducing the risk ofthrombosis in a mammal. Promotion of endogenous thrombolysis is a usefultherapeutic and prophylactic treatment of individual subjects afflictedwith a wide variety of diseases and disorders involving thrombogenesis.Such diseases and disorders include, but are not limited to, heartattack, stroke, deep venous thrombosis, pulmonary embolus, and thepresence in an individual of an abnormally large amount of PAI-1. Thesubject may be any mammal, and is preferably a human subject.

The peptide of the invention may be used alone or in combination withknown thrombolytic agents, thereby permitting the use of far lower andsafer concentrations of known thrombolytic agents. Known thrombolyticagents include, by way of example, tissue plasminogen activator,streptokinase, urokinase, the streptokinase derivative APSAC, andstaphylokinase.

The peptide of the invention may also be used synergistically with knownanticoagulants. The anticoagulants with which the peptide of theinvention may be used include, but are not limited to, those whichinhibit platelet function, those which inhibit the activity of thrombin,those which promote the activity of, for example, activated proteinkinase C, anti-thrombin III, a tissue factor pathway inhibitor, and thelike. Combined use of the peptide of the invention with a knownanticoagulant is effective to prevent thrombus formation and to promotethrombolysis of existing clots.

The peptide of the invention may be used to inhibit PAI-1-dependent celladhesion, a critical control point during cell migration throughtissues. Thus, the peptide of the invention may be used to inhibitangiogenesis, metastasis, the ingrowth of smooth muscle cells intoatherosclerotic plaques, infiltration of leukocytes into inflamed ordamaged tissue, ovulation, binding of spermatozoa to ova, placentaldevelopment, and other types of cell migration, particularly undesirablecell migration.

Table of Abbreviations Used Herein uPA urokinase-type plasminogenactivator scuPA single chain urokinase-type plasminogen activator tcuPAtwo chain urokinase-type plasminogen activator uPAR urokinase-typeplasminogen activator receptor suPAR soluble urokinase-type plasminogenactivator receptor tPA tissue-type plasminogen activator PAI-1plasminogen activator inhibitor-1 α₂MR/LRP α₂-macroglobulin receptor/lowdensity lipoprotein-related receptor protein rRAP recombinant 39kilodalton α₂MR/LRP. PBS phosphate-buffered saline TBS Tris-chloridebuffered saline DMEM Dulbecco's Modified Eagle's Medium BSA bovine serumalbumin.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

The present invention also provides for analogs of the peptidesdescribed herein. Analogs can differ from the peptides described hereinby conservative amino acid sequence differences or by modificationswhich do not affect sequence, or by both.

For example, conservative amino acid changes may be made, which althoughthey alter the primary sequence of the protein or peptide, do notnormally alter its function. Conservative amino acid substitutionstypically include substitutions within the following groups:

glycine, alanine;

valine, isoleucine, leucine;

aspartic acid, glutamic acid;

asparagine, glutamine;

serine, threonine;

lysine, arginine; and

phenylalanine, tyrosine.

Modifications which do not alter the primary sequence of the peptide ofthe invention may be used in the compositions and methods describedherein. Modifications which do not normally alter primary sequenceinclude in vivo, or in vitro chemical derivativization of polypeptides,e.g., acetylation, or carboxylation. Also included are modifications ofglycosylation, e.g., those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps; e.g., by exposing the polypeptide to enzymes whichaffect glycosylation, e.g., mammalian glycosylating or deglycosylatingenzmes. Also embraced are sequences which have phosphorylated amino acidresidues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

Also included are polypeptides which have been modified using ordinarychemical or molecular biological techniques so as to improve theirresistance to proteolytic degradation or to optimize solubilityproperties or to render them more suitable as a pharmaceutical agent.Analogs of such polypeptides include those containing residues otherthan naturally occurring L-amino acids, e.g., D-amino acids ornon-naturally occurring synthetic amino acids. The peptides of theinvention are not limited to products of any of the specific exemplaryprocesses listed herein.

The peptide of the invention has the general formula

X₁X₂X₃X₄X₅X₆X₇X₈

wherein:

X₁ is hydrogen, an amino-terminal blocking group, or one to twenty aminoacid residues;

X₂ is an amino acid selected from the group consisting of D, E, H, K,and R;

X₃ is an amino acid selected from the group consisting of E and D;

X₄ is an amino acid selected from the group consisting of I, L, and V;

X₅ is an amino acid selected from the group consisting of I, L, and V;

X₆ is an amino acid selected from the group consisting of M;

X₇ is an amino acid selected from the group consisting of D, E, H, K,and R; and

X₈ is hydrogen, a carboxyl-terminal blocking group, or one to twentyamino acid residues.

Preferably, X₁ is hydrogen or an amino-terminal blocking group, X₂ is anamino acid selected from the group consisting of D, E, and R, X₃ is anamino acid selected from the group consisting of D and E, X₄ is I, X₅ isI, X₆ is M, X₇ is an amino acid selected from the group consisting of Dand E, and X₈ is hydrogen or a carboxyl-terminal blocking group. Morepreferably, X₁ is hydrogen, X₂ is E, X₃ is E, X₄ is I, X₅ is I, X₆ is M,X₇ is D, and X₈ is hydrogen.

Most preferably, the peptide of the invention is EEIIMD.

The peptide of the invention is also referred to herein as the “PAI-1”peptide.

The invention encompasses the preparation and use of pharmaceuticalcompositions comprising the peptide of the invention as an activeingredient. Such a pharmaceutical composition may consist of the activeingredient alone, in a form suitable for administration to a subject, orthe pharmaceutical composition may comprise the active ingredient andone or more pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. Administration of one ofthese pharmaceutical compositions to a subject is useful for treating avariety of diseases or disorders as described elsewhere herein. Theactive ingredient may be present in the pharmaceutical composition inthe form of a physiologically acceptable ester or salt, such as incombination with a physiologically acceptable cation or anion, as iswell known in the art.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs,birds including commercially relevant birds such as chickens, ducks,geese, and turkeys, fish including farm-raised fish and aquarium fish,and crustaceans such as farm-raised shellfish.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, in asal,buccal, ophthalmic, or another route of administration. Othercontemplated formulations include projected nanoparticles, liposomalpreparations, resealed erythrocytes containing the active ingredient,and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A tablet-comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid,and sorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e. about 20° C.) and which isliquid at the rectal temperature of the subject (i.e. about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for vaginal administration. Such acomposition may be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or a solution for vaginal irrigation.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e. such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made bycombining the active ingredient with a pharmaceutically acceptableliquid carrier. As is well known in the art, douche preparations may beadministered using, and may be packaged within, a delivery deviceadapted to the vaginal anatomy of the subject. Douche preparations mayfurther comprise various additional ingredients including, but notlimited to, antioxidants, antibiotics, antifungal agents, andpreservatives.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and preferably from about 1 toabout 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers. Such a formulation is administered inthe manner in which snuff is taken i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient. Suchpowdered, aerosolized, or aerosolized formulations, when dispersed,preferably have an average particle or droplet size in the range fromabout 0.1 to about 200 nanometers, and may further comprise one or moreof the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other ophthalmalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

Typically dosages of the peptide of the invention which may beadministered to an animal, preferably a human, range in amount from 1 μgto about 100 g per killogram of body weight of the animal. While theprecise dosage administered will vary depending upon any number offactors, including but not limited to, the type of animal and type ofdisease state being treated, the age of the animal and the route ofadministration. Preferably, the dosage of the compound will vary fromabout 1 mg to about 10 g per killogram of body weight of the animal.More preferably, the dosage will vary from about 10 mg to about 1 g perkillogram of body weight of the animal.

The compound may be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even leesfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the animal, etc.

The invention also includes a kit comprising the peptide of theinvention and an instructional material which describes adventitiallyadministering the peptide to a cell or a tissue of a mammal. In anotherembodiment, this kit comprises a (preferably sterile) solvent suitablefor dissolving or suspending the peptide of the invention prior toadministering the compound to the mammal.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the peptide of the invention inthe kit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of alleviation the diseases ordisorders in a cell or a tissue of a mammal. The instructional materialof the kit of the invention may, for example, be affixed to a containerwhich contains peptide of the invention or be shipped together with acontainer which contains the peptide. Alternatively, the instructionalmaterial may be shipped separately from the container with the intentionthat the instructional material and the compound be used cooperativelyby the recipient.

The invention is now described with reference to the followingexperimental details. The experimental details are provided for thepurpose of illustration only and the invention should in no way beconstrued as being limited to the embodiments described herein, butrather should be construed to encompass any and all variations whichbecome evident as a result of the teaching provided herein.

EXAMPLE 1

Regulation of scuPA by the Peptide of the Invention

To examine the mechanism by which uPA binds to α₂MR/LRP, the effect ofthe presence of peptide EEIIMD (SEQ ID NO: 3) on the cellular binding,interalization, and degradation of scuPA, the scuPA-suPAR complex, andtcuPA was examined. The peptide increased recognition of scuPA, but nottcuPA, by α₂MR/LRP. The data indicate that interaction of scuPA with aportion of PAI-1, results in a conformational change of scuPA thatmodulates the activity thereof. The peptide of the invention wasdiscovered to promote internalization and degradation of scuPA by cellswhich express α₂MR/LRP.

The materials and methods used in the experiments of this Example arenow described.

Recombinant scuPA and recombinant suPAR were purified as reported andwere obtained from Abbott Laboratories (Abbott Park, Ill.; Higazi etal., 1996, Blood 87:3545-3549; Higazi et al., 1997, J. Biol. Chem.272:5348-5353). Peptides EEIIMD (SEQ ID NO: 3), REIIMD (SEQ ID NO: 4),and EEIIMR (SEQ ID NO: 5) were synthesized using methods well known inthe art of peptide synthesis. Glu-plasminogen, tcuPA, PAI-1, and theplasmin chromogenic substrate Spectrazyme PLT were obtained fromAmerican Diagnostica, Inc. (Greenwich, Conn.). Purified a₂MR/LRP andrRAP were prepared as described (Williams et al., 1992, J. Biol. Chem.267:9035-9040). The LM-TK⁻ cell line was obtained from the American TypeTissue Collection (catalog number CCL 1.3 L-M(TK⁻) Rockville, Md.).scuPA, suPAR, and tcuPA were radiolabeled with ¹²⁵I as described(Bamathan et al., 1990, J. Biol. Chem. 265:2865-2872) using iodobeadsobtained from the Pierce Chemical Company (Rockford, Ill.).

Preparation of Protein Complexes

To prepare the scuPA-suPAR complex, suPAR and scuPA were incubatedtogether at a molar ratio of 1.25 to 1, respectively, for one hour at37° C. at ten times the desired final concentration in binding buffer(comprising PBS supplemented with 1.5% (w/v) BSA).

To form complexes with PAI-1, PAI-1 was added to a solution comprisingbinding buffer and scuPA, tcuPA, the scuPA/suPAR complex, or thetcuPA/suPAR complex at a 1:1 molar ratio, and the solution was incubatedfor thirty minutes at 37° C.

Protein complexes were diluted to a desired working concentrationimmediately before use.

Assessment of Plasminogen Activator Activity

A solution comprising 5 nanomolar tcuPA, 0 or 200 micromolar peptideEEIIMD (SEQ ID NO:3), and 0 or 200 micromolar peptide REIIMD (SEQ IDNO:4) was incubated for thirty minutes. The solution was then added to areaction mixture comprising 25 nanomolar PAI-1, 50 nanomolarGlu-plasminogen, and 50 micromolar chromogenic substrate, and theoptical density of the mixture at 405 nanometers was measuredcontinuously, as described (Higazi et al., 1996, Blood 87:3545-3549).

Lipand Binding Assays

Binding of radiolabeled ligands, including scuPA and tcuPA, to cells wasmeasured as described (Higazi et al., 1996, Blood 88:542-551). Briefly,LM-TK⁻ cells were suspended in DMEM (GIBCO, Grand Island, N.Y.)containing 10% (v/v) fetal calf serum and were grown to confluence at37° C. overnight in 96-well Falcon™ multiwell tissue culture dishes(Becton Dickinson, Lincoln Park, N.J.). The cells were chilled for onehour on ice and were washed twice with chilled binding buffer.¹²⁵I-labeled ligands, with or without 50-fold molar excess ofnon-labeled ligands, were added to the cells in the presence or absenceof 0-300 micromolar peptide, and the cell cultures were incubated fortwo hours at 4° C. The cells were washed four times with binding buffer,solubilized in 0.1 N NaOH, and the cell extract was assessed forradioactivity. In other experiments, binding of labeled ligands wasperformed in the absence or presence of 400 nanomolar rRAP diluted inTBS containing 4 millimolar Ca²⁺, or in the presence of 100 microgramsper milliliter affinity purified IgG anti-α₂MR/LRP antibody. Assays wererepeated at least three times, and data presented herein represent themean and standard deviation.

Solid Phase Bindings Assay

To measure the binding of labeled ligands to α₂MR/LRP, a 96-wellmicrotiter plate was incubated with 3 micrograms per milliliter purifiedα₂MR/LRP or with 3 micrograms per milliliter BSA in TBS containing 4millimolar Ca²⁺ overnight at 4° C. The buffer was removed, and thenon-reacted sites on the plate were blocked using a blocking solutioncomprising TBS, 4 millimolar Ca²⁺, 0.05% (v/v) Tween-20, and 3% (w/v)BSA. The wells of the plate were filled with the blocking solution andincubated for one hour at 4° C. Next, the blocking solution was removed,each well was filled with a solution comprising blocking solution and 0or 200 nanomolar rRAP, and the plate was incubated at 4° C. for anotherhour. Binding of ¹²⁵I-labeled ligands to immobilized α₂MR/LRP wasdetermined as described (Higazi et al., 1996, Blood 88:542-551; Nykjaeret al., 1993, J. Biol. Chem. 268:15048-15055).

Assessment of Internalization and Degradation of scuPA by LM-TK⁻ Cells

LM-TK⁻ cells were grown overnight at 37° C. to confluence in 48-wellFalcon™ Multiwell tissue culture dishes (Becton Dickinson, Lincoln Park,N.J.). Cells were prechilled on ice for one hour, washed twice with abuffer comprising TBS, 4 millimolar Ca²⁺, and 3% (w/v) BSA, andincubated for one hour at room temperature with the same buffer or withbuffer supplemented with either 400 nanomolar rRAP or 100 micrograms permilliliter IgG anti-α₂MR/LRP. The buffer was removed, 125I-scuPA wasadded to the cells, and the cells were incubated for two hours at 4° C.in the presence of 0-300 micromolar peptide, 0 or 400 nanomolar rRAP,and 0 or 100 micrograms per milliliter anti-α₂MR/LRP. Unbound ligand wasremoved and cells were washed five times with the buffer. DMEMcomprising 4 millimolar Ca²⁺, 0 or 400 nanomolar rRAP, and 0 or 100micrograms per milliliter anti-α₂MR/LRP was added to the cells, and thecells were incubated for eighteen hours at 37° C. Internalization anddegradation of scuPA were measured as described (Kounnas et al., 1993,J. Biol. Chem. 268:21862-21867; Li et al., 1994, J. Biol. Chem.269:8153-8158). Briefly, to measure internalization, cells were washedtwice with the buffer, a solution comprising 50 millimolar glycine 150millimolar NaCl at pH 3.0 was added to the cells, and the cells wereincubated for fifteen minutes at 4° C. to dissociate cell surface-boundligands. Cells were solubilized by adding 0.1 N NaOH to thecell-containing solution and incubating the solution for ten minutes.Radioactivity in the cell extract was assessed. To measure scuPAdegradation, the medium was removed from each cell culture after theeighteen hour incubation period, trichloroacetic acid was added to afinal concentration of 10% (v/v), precipitated protein was separated bycentrifugation, and radioactivity in the supernatant was assessed.

The results of the experiments presented in this Example are nowdescribed.

Peptide EEIIMD (SEQ ID NO: 3) promoted binding of scuPA to LM-TK⁻ cells.The data presented herein in FIG. 1A indicate that only a minimal amountof scuPA bound to LM-TK⁻ cells, consistent with a previous report(Higazi et al., 1996, Blood 88:542-551) when scuPA was present in theextracellular medium. Binding of scuPA to these cells was promoted bythe presence of peptide EEIIMD in a dose-dependent and saturable manner,both with respect to scuPA concentration, as depicted in FIG. 1A, andwith respect to peptide concentration, as depicted in FIG. 1B.

Promotion of scuPA binding to LM-TK⁻ cells was evident at allconcentrations of scuPA tested, and the increase in scuPA binding in thepresence of peptide EEIIMD (SEQ ID NO: 3) was primarily the result of afour-fold increase in the value of Bmax. Using Scatchard analysis, itwas determined that little specific binding of scuPA to LM-TK⁻ cellsoccurred in the absence of peptide EEIIMD, the value of K_(d) for scuPAbeing greater than one micromolar in the absence of the peptide. Incontrast, the value of K_(d) for scuPA in the presence of the peptidewas approximately 35 nanomolar. Half maximal stimulation of scuPAbinding to LM-TK⁻ cells was achieved using a peptide EEIIMDconcentration equal to approximately 50 micromolar, the near-maximaleffect being observed using approximately 300 micromolar peptide, asdepicted in FIG. 1B.

Studies were performed to identify the sequence requirements of thepeptide of the invention involved in stimulation of scuPA binding toLM-TK⁻ cells. Peptide EEIIMD (SEQ Id NO: 3) was the lead compound inthese investigations. Two peptides, having amino acid sequences REIIMD(SEQ ID NO:4) and EEIIMR, were prepared, and the effect of the presenceof these peptides on the binding of scuPA to LM-TK⁻ cells wasinvestigated, analogously to the investigations of the effect of thepresence of EEIIMD described herein. As depicted in FIG. 1A, it wasfound that peptide REIIMD was nearly as potent a promoter of scuPAbinding to LM-TK⁻ cells as was peptide EEIIMD. In contrast, peptideEEIIMR exhibited approximately 20% of the scuPA-binding-promotingactivity exhibited by peptide EEIIMD.

Hence, it is apparent that the charge of the amino acid residue at theamino terminus of peptide EEIIMD (SEQ ID NO: 3) is not important.Substitution of this Glu residue by Arg has been demonstrated to resultin a peptide which is an effective promoter of scuPA binding to LM-TK⁻cells. Therefore, the amino terminal Glu residue of peptide EEIIMD maybe substituted with Arg, His, Lys, or Asp to generate peptides which areincluded in the invention. Thus, peptides having the general formulaXEIIMD may be used as effective promoters of scuPA binding to LM-TK⁻cells, wherein X is an amino acid residue selected from the groupconsisting of D, E, H, K, and R. Preferably, X is D, E, or R. Alsopreferably, X is D or E. Most preferably, X is E.

Furthermore, it is apparent that the charge of the amino acid residue atthe carboxyl terminus of peptide EEIIMD (SEQ ID NO:3) is import to themagnitude, but not to the existence of the capacity of the peptide topromote scuPA binding to LM-TK⁻ cells. It has been demonstrated hereinthat if the carboxyl terminal amino acid residue has a positive charge,the peptide will have a reduced, but non-negligible, capacity to promotescuPA binding to LM-TK⁻ cells, relative to peptide EEIIMD. Thus,peptides having the general formula EEIIMD are included in the inventionand may be used as effective promoters of scuPA binding to LM-TK⁻ cells,wherein Z is an amino acid residue selected from the group consisting ofD, E, H, K, and R. Preferably, Z is D, E, or R. Also preferably, Z is Dor E. Most preferably, Z is E.

It has been reported that substitution of the glutamic acid residue atamino acid position 350 of PAI-1 with an arginine residue does notaffect the capacity of PAI-1 to inhibit the proteolytic activity of tPA,and that substitution of the aspartic acid residue at amino acidposition 355 of PAI-1 with an arginine residue prevents PAI-1 frominhibiting the proteolytic activity of tPA (Madison et al., 1990, J.Biol. Chem. 265:21423-21426). The report of Madison et al. is consistentwith the results described herein, even though Madison et al. studiedthe interaction of PAI-1 with a different protein, namely tPA, and usedfull-length PAI-1 protein, rather than the novel peptide of theinvention.

The effect of the presence of peptide EEIIMD (SEQ ID NO:3) on binding ofscuPA to LM-TK⁻ cells in the presence of PAI-1 was examined.Preincubation of scuPA with PAI-1 prior to addition of peptide EEIIMDminimally increased binding of ¹²⁵I-scuPA to LM-TK⁻ cells, as depictedin FIG. 2. This observation is consistent with the low affinity of PAI-1for scuPA that has been reported (Kruithof, 1988, Enzyme 40:113-121;Lijnen et al., 1994, Eur. J. Biochem. 224:567-574; Kruithof et al.,1984, Blood 64:907-913; Andreasen et al., 1986, J. Biol. Chem.261:7644-7651; Manchanda et al., 1995, J. Biol. Chem. 270:20032-20035).The scuPA-binding-promoting effect of peptide EEIIMD in the presence ofPAI-1 was identical to the effect observed in the absence of PAI-1.Hence, it is clear that PAI-1 did not displace the peptide from Theeffect of PAI-1 and peptide EEIIMD (SEQ ID NO:3) on the binding of thescuPA-suPAR complex and of tcuPA to LM-TK⁻ cells was examined. Asdepicted in FIG. 3, the presences of PAI-1 and peptide EEIIMD eachcaused a minimal increase in binding of the scuPA-suPAR complex toLM-TK⁻ cells. PAI-1 clearly stimulated binding of tcuPA, as reported byothers (Nykjaer et al., 1994, J. Biol. Chem. 269:25668-25676; Nykjaer etal., 1992,J. Biol. Chem. 267:14543-14546). In contrast, as depicted inFIG. 3, peptide EEIIMD had no effect on binding of the tcuPA-suPARcomplex to LM-TK⁻ cells. The presence of peptide EEIIMD also failed tostimulate binding of tcuPA to LM-TK⁻ cells. The failure of peptideEEIIMD to stimulate binding of tcuPA or the tcuPA-suPAR complex toLM-TK⁻ or to more than minimally stimulate binding of the scuPA-suPARcomplex to LM-TK⁻ cells likely results from either the inability of thepeptide to bind to the site of the scuPA protein to which PAI-1 binds orthe conformational state of scuPA. To address the question of whetherpeptide EEIIMD can bind to the site of the uPA protein to which PAI-1binds, the capacity of the peptide to competitively inhibit PAI-1activity was assessed using tcuPA as the substrate of PAI-1. The datadepicted in FIG. 4 indicate that peptide EEIIMD inhibits PAI-1 activity.The control peptide EEIIMR did not inhibit PAI-1 activity. Hence,peptide EEIIMD and PAI-1 appear to interact with at least one commonportion of scuPA.

It is understood that complexes between tcuPA and PAI-1 are internalizedand degraded following binding of the tcuPA-PAI-1 complex to α₂MR/LRP(Li et al., 1994, J. Biol. Chem. 269:8153-8158; Nykjaer et al., 1992, J.Biol. Chem. 267:14543-14546). Investigations were performed to determinewhether the presence of peptide EEIIMD increased the binding,internalization, and degradation of scuPA through this pathway.

Peptide EEIIMD (SEQ ID NO:3) promoted binding of scuPA to purifiedα₂MR/LRP, and the promoting effect of peptide EEIIMD was inhibited bythe presence rRAP, as depicted in FIG. 5A. In addition, promotion bypeptide EEIIMD of binding of scupA to LM-TK⁻ cells was inhibitedapproximately 70% by the presence of 400 nanomolar rRAP andapproximately 85% by the presence of affinity purified anti-α₂MR/LRPIgG, as depicted in FIG. 5B. Furthermore, EEIIMD promoted bothinternalization and degradation of scuPA by LM-TK⁻ cells, as depicted inFIG. 6. Promotion of scuPA internalization and degradation by LM-TK⁻cells was inhibited approximately 70% by the presence of 400 nanomolarrRAP and approximately 80% by the presence 100 micrograms per milliliterof anti-α₂MR/LRP IgG.

Although it has been postulated that binding of the tcuPA-PAI-1 complexto α₂MR/LRP is mediated by independent epitopes in tcuPA and α₂MR/LRP(Nykjaer et al., 1994, J. Biol. Chem. 269:25668-25676), such a mechanismdoes not explain the stimulatory effect of the presence of peptideEEIIMD (SEQ ID NO: 3) on scuPA binding to α₂MR/LRP which has beendescribed herein. The peptide of the invention is not large enough toaffect both scuPA and α₂MR/LRP simultaneously. Furthermore, peptideEEIIMD bound to tcuPA, as shown by inhibition of PAI-1 activity, but didnot increase tcuPA binding to α₂MR/LRP. Therefore, the observation thatthe presence of peptide EEIIMD increased the value of B_(max) forbinding of scuPA to α₂MR/LRP suggests that the peptide induces apreviously unrecognized alteration in the secondary structure of scuPA,relative the conformational state of the native molecule, that isspecifically recognized by α₂MR/LRP. Put another way, the resultsdescribed herein suggest that the site on the scuPA protein to whichpeptide EEIIMD binds functions as an “allosteric” site. Consistent withthis notion, the presence of peptide EEIIMD stimulated binding of scuPAto α₂MR/LRP, but had no effect on binding of either the scuPA-suPARcomplex or tcuPA to α₂MR/LRP. These results are consistent with thereported differences in the ability of scuPA, the scuPA-suPAR complexand tcuPA to interact with α₂MR/LRP (Nykjaer et al., 1994, J. Biol.Chem. 269:25668-25676).

It is likely that peptide EEIIMD (SEQ ID NO: 3) fails to stimulatecellular binding of tcuPA, even though this peptide binds to tcuPA,because the binding of peptide EEIIMD to tcuPA does not induce tcuPA toassume a secondary structure including the site recognized by α₂MR/LRP.A similar mechanism may also account for the failure of peptide EEIIMDto induce cellular binding of the scuPA-suPAR complex to α₂MR/LRP.Alternatively, the binding site for α₂MR/LRP on scuPA may be induced andshielded by uPAR, consistent with the capacity of uPAR to blockinteraction of scuPA and the tcuPA-PAI-1 complex with α₂MR/LRP (Nykjaeret al., 1994, J. Biol. Chem. 269:25668-25676; Higazi et al., 1996, Blood88:542-551). Thus, conversion of scuPA to tcuPA is associated with theloss of the ability of peptide EEIIMD to induce the structure recognizedby α₂MR/LRP, presumably as a result of the loss of coordinateinteraction between different portions of the molecule. Support for thisinterpretation comes from the observations that uPAR has little or noeffect on the enzymatic activity of tcuPA (Higazi et al., 1995, J. Biol.Chem. 270:17375-17380) or on its susceptibility to inactivation by PAI-1(Higazi et al., 1995, J. Biol. Chem. 270:17375-17380; Ellis et al.,1990, J. Biol. Chem. 265:9904-9908). These results provide additionalsupport for the idea that the conversion of scuPA to tcuPA is the firststep in the inactivation and degradation of scuPA.

Irrespective of the sequence of events, the capacity of the presence ofpeptide EEIIMD (SEQ ID NO: 3) to stimulate cellular binding of scuPAprovides additional support for the notion that the physiologicalactivity of scuPA can be regulated by altering its conformation, as aresult of which internalization and degradation of the protein isaccelerated. Thus, using the compositions and methods described herein,scuPA can be rendered susceptible to binding by α₂MR/LRP without theneed to first convert scuPA to tcuPA. No physiological analogue of theactivity exhibited by peptide EEIIMD, as described herein, has beenreported.

EXAMPLE 2

Models for Testing the Potency of PAI-1 peptide in vivo

Three models have been developed to evaluate the PAI-1 peptide of theinvention in vivo.

1) Effect of PAI-1 Peptide on uPA Mediated-fibrinolysis in vivo

An in vivo model of clot lysis was developed. In this model, ahomogenous preparation of radiolabeled micro-emboli was injected intothe tail vein of a mouse. The spontaneous and uPA-mediated clearance ofthe clots over time was then assessed.

The Materials and Methods used in the experiments presented in thisExample are now described.

Preparation of Micro-Emboli

Plasminogen-depleted human fibrinogen was radiolabeled with ¹²⁵1 usingIodo-beads and the free iodine was removed using a PD-10 column(Pharmacia, Piscataway, N.J.) ¹²⁵I-fibrinogen (−40×10⁶ cpm) was added to1.2 mL of unlabeled fibrinogen (35 mg/mL final concentration) prior toforming micro-emboli. To form the clot, whole blood from a healthyvolunteer was collected in citrate (0.32% final concentration). Plasmawas isolated by centrifugation at 1200×g. Plasma (2.5 mL) was mixed with0.1 mL trace labeled ¹²⁵ 1-human fibrinogen (40 mg; 10 mg/mL finalconcentration in a glass tube. CaCl₂ (20 mM final concentration) andhuman thrombin (Sigma; 0.2 U/mL, final concentration) were added at roomtemperature for 1 hour and maintained overnight at 4° C. All subsequentsteps were performed at 4° C. as well. The clots were decanted onplastic lids, cut into small pieces and resuspended in 2 mL Kreb'sRinger's buffer (KRB). The clots were homogenized for 30 seconds using aPT-10/35 Polytron homogenizer (Brinkmann Instr., Westbury, N.Y.) atmid-speed. After homogenization, the samples were centrifuged at 2000×gfor 15 min. The supernatant was removed and the pellet was resuspendedin KRB. A second homogenization was performed at higher speed for 30seconds and the pellet was washed as described. After the finalhomogenization, the micro-emboli were suspended in 13 mL KRB-bovineserum albumin (BSA) (3 mg/mL). ¹²⁵I-micro-emboli aliquots were stored at4° C. and were used within 24 hour. The preparation was mixed byrepetitive pipetting and allowed to sediment for 5 minutes just prior touse to eliminate any larger particles which remained. The supernatantwas split into 200 μl aliquots immediately prior to injection. Randomaliquots were selected to characterize the size distribution of themicro-emboli using a ZM Coulter counter (Coulter Electronics, LTD.Hialeah, Fla.).

Mice

Mice having genetic deletions in tissue-type plasminogen activator(tPA^(−/−)), urokinase-type plasminogen activator (uPA^(−/−)), and theurokinase receptor (uPAR^(−/−)), each on a 25% Swiss/75%C57/blackbackground and their respective littermate controls were used (Carnelietet al., 1994, Nature 369:419_(—)424; Dwerchin et al., 1996, J. Clin.Invest. 97:870-878). tPA^(−/−) on a C57/black background wild typeC57/black mice and wild type Balb/c mice were obtained from JacksonLaboratories (Bar Harbor, Me.). All of the mice weighed 20-30 g at thetime of study. There was no influence of the various genetic backgroundsof the wild type mice with respect to endogenous fibrinolysis.

Plasma Clearance

¹²⁵I-micro-emboli (200 μl aliquots containing 15-30,000 cpm) wereresuspended by pipetting several times immediately before loading a 21gauge syringe. Mice were injected via the tail vein and returned totheir cages until sacrifice. At various times after injection (10minutes, 1, 3 and 5 hours), the mice were anesthetized using metofane,and 100 μl of blood was withdrawn by retroorbital puncture into aheparinized capillary pipette. The mice were sacrificed by cervicaldislocation. The major organs were harvested immediately, rinsed insaline, dried on Whatman paper and the radioactivity in each tissue wasmeasured. The exact dose (cpm) injected into each mouse was calculatedby subtracting the residual radioactivity remaining in the tube andsyringe after injection. Radioactivity in the tail of each mouse wascounted to verify that the injection was complete. In some experiments,the lungs were obtained at 10 minutes after injection and were fixed informalin for immunohistochernical staining. In another set ofexperiments, the lungs were exposed to X-ray film to determine thedistribution of radioactivity. In a third series of experiments, theindividual lobes from each lung were isolated and the radioactivitytherein was assessed.

Recovery Experiments

Two chain urokinase (tcuPA) was obtained from American Diagnostica(Greenwich, Conn.). uPA^(−/−) mice were anesthetized by intraperitonealinjection of Nembutal (50 mg/kg). A polyethylene catheter, siliconizedwith Sigmacot solution (Sigma Chemical Co. St. Louis, Mo.), was washedwith PBS and cannulated in the jugular vein. tcuPA or PBS was infusedusing a PHD 2000 multi-syringe pump at a rate of 15 μL/min for the first5 minutes and then 5 μL/minute for 60 minutes. The ¹²⁵1-micro-emboliwere injected into the tail vein as described above 5 minutes after theinfusion was started. The mice were kept under anesthesia throughout theentire experiment. At the completion of the infusion, the mice weresacrificed and the tissues were harvested and counted.

The Results of the experiments presented in this Example are nowdescribed.

In FIG. 7 there is shown a comparison of the distribution of labeledfibrin micro-emboli and radiolabeled fibrinogen. The data illustratethat whereas fibrinogen was observed predominantly in the blood, thefibrin clots localized primarily within the lung. This was confirmed byautoradiography of the lungs and by immunohistochemical staining. Bothassays demonstrated a homogeneous distribution of radiolabeled fibrinclots in the microvasculature through all lobes.

In FIG. 8, there is shown the clearance of the micro-emboli from thelungs of wild type animals and animals having various deletion mutationsin tissue-type plasminogen activator (tPA^(−/−)), urokinase-typeplasminogen activator (uPA^(−/−)), and the urokinase receptor(uPAR^(−/−)). The data clearly demonstrate that fibrinolysis wasimpaired in mice lacking either urokinase or tPA. This is the firstknown demonstration of a defect in endogenous pulmonary fibrinolysis inuPA^(−/−) mice.

In FIG. 9, there is shown the results when the phenotype of theuPA^(−/−) mice was rescued by infusion of two chain uPA. Clot lysis inthe uPA^(−/−) mice was complete.

This model provides a rapid, i.e., one hour, non-lethal, reproducible,biologically relevant, scuPA-dependent endpoint in a small animal modelusing human clots.

To determine whether the PAI-I peptide act synergistically with tcuPA topromote fibrinolysis, competition experiments using endogenous PAI-1 maybe performed. Experiments are conducted in wild type animals byinvesting the clot and/or the mouse plasma with murine or human PAI-1and in mice which overexpressing human PAI-I (Eitzman et al., 1996, J.Clin. Invest. 97:232-237). To conduct such experiments, dose-responseexperiments are performed using PAI-I peptide and a scrambled peptidecontrol in the presence of a constant amount of tcuPA and PAI-1. Thepeptide is infused immediately before or coincident with tcuPA. Theclearance of ¹²⁵1-tcuPA may also be measured in the presence of PAI-Ipeptide. It is hypothesized that this clearance will be impeded sincethe major pathway for the removal of ¹²⁵1-tcuPA is mediated bydeterminants on PAI-I. It is also hypothesized that the PAI-I peptideshould dramatically lessen the amount of urokinase required for clotlysis. In addition, it has recently been reported that clot-boundvitronectin binds and stabilizes the PAI-1 in an active conformation.Therefore, experiments which are designed to determine whether the PAI-Ipeptide competes for vitronectin binding thereby promoting fibrinolysisby removing active inhibitor from the clot may be performed.

2) The Effect of the PAI-I Peptide on Tumor Cell Adhesion and MetastasisFormation

PAI-1 plays an important role in regulating in tumor cell adhesion andthereby regulating tumor metastasis. There is evidence to suggest thatthis effect is mediated by competition between PAI-I and vironectin forthe urokinase receptor, an important mediator of certain β-integrins(Stefansson et al., 1996, Nature 383:441-443; Deng et al., 1996, J. CellBiol. 134:1563-1571). Recent data indicates that mice with a targeteddeletion in PAI-1 exhibit fewer metastases as a result of impaired tumoradhesion (Bajou et al., 1998, Nature Med. 4:923-928). It is hypothesizedthat the PAI-1 peptide will compete with native PAI-I thereby having ananti-metastatic effect on cells. Further, as the data presented hereinestablish, the PAI-I peptide accelerates the internalization of uPA bya₂MR/LRP which represents a second locus of anti-metastatic activity.

Experiments may be directed to examining the effect of PAI-I peptide oncell adhesion in vitro. The adhesion of U937 cells may be measured by aminor modification of the method described by Li et al. (1995, J. Biol.Chem. 270:30282-30285). Briefly, U937 cells stimulated with TNFα: andlabeled with ³H-thymidine are added to vitronectin-pretreated oruntreated plastic wells in the presence of scuPA (1 nM). To study theanti-adhesive effects, the PAI-1 derived peptide (0-100 μM) or ascrambled peptide control is added contemporaneously with uPA. Atvarious times, the wells are washed, the cells are lysed and thereleased radioactivity is determined. The potency of the anti-adhesiveeffect is based on a comparison of the IC50 versus 1 nM scuPA.

As a second step, the effect of the PAI-1 peptide on the migration ofendothelial cells through collagen coated membranes as a model ofpathologic angiogenesis may be examined. Receptor-associated scuPA maybe responsible for urokinase activity on endothelial cells and on othercell types (Barnathan et al., 1990, J. Biol. Chem. 265:2865-2872;Manchanda et al., 1991, J. Biol. Chem. 266:14580-14584). The datapresented herein have established that PAI-I peptide promotes scuPAclearance and competes with the pro-adhesive effects of PAI-1.Therefore, this model is effective to test both the anti-adhesive andanti-proteolytic effects of the PAI-1 peptide.

In a third step, the anti-metastatic capacity of the PAI-1 peptide invivo may be demonstrated. To accomplish this, stable tumor cell linesthat express uPA which is PAI-1 resistant have been generated.Specifically, a syngeneic cell line has been developed that coexpresseshuman uPAR and a plasmin-insensitive scuPA (scuPA-glu¹⁵⁸). This mutationprevents conversion of uPA to two chain urokinase and thereby assuresthat any biological activity of this molecule is due to receptor boundand activated single chain uPA. Data have been obtained which establishthat scuPA-glu¹⁵⁸ bound to uPAR is enzymatically active and insensitiveto PAI-I in vitro. The tumor cells are injected into syngeneic rats inthe presence/absence of PAI-1 peptide or a scrambled peptide control.The growth of the primary tumor and formation of pulmonary metastases ismonitored over the subsequent fourteen days. It is predicted that thesetumor cells will metastasize readily in the absence of PAI-I peptide butthat the peptide will inhibit both their local growth and metastaticcapacity through an effect on uPA-mediated cell adhesion andproteolysis.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be advised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

5 1 6 PRT Artificial Sequence Description of Artificial SequenceResidues 179-184 of urokinase-type plasminogen activator 1 Arg His ArgGly Gly Ser 1 5 2 11 PRT Artificial Sequence Description of ArtificialSequence Residues 346-356 of plasminogen activator inhibitor-1 2 Arg MetAla Pro Glu Glu Ile Ile Met Asp Arg 1 5 10 3 6 PRT Artificial SequenceDescription of Artificial Sequence Peptide for modulating binding ofscuPA with LM-TK(-) cells 3 Glu Glu Ile Ile Met Asp 1 5 4 6 PRTArtificial Sequence Description of Artificial Sequence Peptide formodulating binding of scuPA with LM-TK(-) cells 4 Arg Glu Ile Ile MetAsp 1 5 5 6 PRT Artificial Sequence Description of Artificial SequencePeptide for modulating binding of scuPA with LM-TK(-) cells 5 Glu GluIle Ile Met Arg 1 5

What is claimed is:
 1. A composition comprising a peptide consisting of the amino acid sequence X₁X₂X₃X₄X₅X₆X₇X₈, wherein: X₁ is hydrogen or an amino-terminal blocking group; X₂ is an amino acid selected from the group consisting of D, E, and R; X₃ is an amino acid selected from the group consisting of D and E; X₄ is I; X₅ is I; X₆is M; X₇ is an amino acid selected from the group consisting of D and E; and X₈ is hydrogen or a carboxyl-terminal blocking group.
 2. A composition comprising a peptide consisting of the amino acid sequence X₁X₂X₃X₄X₅X₆X₇X₈, wherein: X₁ is hydrogen; X₂ is E; X₃ is E; X₄ is I; X₅ is I; X₆ is M; X₇ is D; and X₈ is hydrogen.
 3. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.
 4. A kit comprising a peptide having the amino acid sequence X₁X₂X₃X₄X₅X₆X₇X₈, wherein: X₁ is hydrogen or an amino-terminal blocking group; X₂ is an amino acid selected from the group consisting of D, E, and R; X₃ is an amino acid selected from the group consisting of D and E; X₄ is I; X, is I; X₆ is M; X₇ is an amino acid selected from the group consisting of D, E, H, K, and R; and X₈ is hydrogen or a carboxyl-terminal blocking group; and an instructional material for using the kit.
 5. The composition of claim 2, further comprising a pharmaceutically acceptable carrier. 